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MicroRNA expression profiles in Aromatase Inhibitor-Resistant, Tamoxifen-Resistant and LTED breast cancer cell lines

ABSTRACT: Resistance to endocrine therapy agents has presented a clinical obstacle in the treatment of hormone-dependent breast cancer. Our laboratory has initiated a study of microRNA regulation of signaling pathways that may result in breast cancer progression on aromatase inhibitors (AI). Microarray analysis of microRNA expression identified 115 significantly regulated microRNAs, of which 49 microRNAs were believed to be hormone-responsive. Within the AI-resistant cells, microRNAs were differentially expressed between the steroidal and non-steroidal AI-resistant lines. Also, a group of microRNAs were inversely expressed in the AI-resistant lines versus LTEDaro and tamoxifen-resistant. We focused our work on hsa-miR-128a which was hormone-responsive and up-regulated in the letrozole-resistant cell lines. Human miR-128a was shown to negatively target TGFBRI protein expression by binding to the 3’UTR region of the gene. Loss of TGFBRI resulted in compromised sensitivity to the growth inhibitory effects of TGFB in the letrozole-resistant lines. Inhibition of endogenous miR-128a resulted in re-sensitization of the letrozole-resistant lines to TGFB growth inhibitory effects. This data suggests that the hormone-responsive miR-128a can modulate TGFB signaling and survival of the letrozole-resistant cell lines. To our knowledge, this is the first study to address the role of microRNA regulation as well as TGFB signaling in AI-resistant breast cancer cell lines. We believe that in addition to estrogen-modulation of gene expression, hormone-regulated microRNAs may provide an additional level of post-transcriptional regulation of signaling pathways critically involved in breast cancer progression and AI-resistance. To look at microRNA expression profiles of breast cancer cell lines derived from MCF-7 cells that are resistant to endocrine therapy agents. MCF-7 cells that overexpress aromatase (MCF-7aro) were cultured long-term in the presence of endocrine therapy agents until cells acquired resistance. Three different aromatase inhibitors (letrozole, anastrozole or exemestane) were used, as well as the ER antagonist tamoxifen, or the hormone-free long-term estrogen deprived cells (LTED). Three replicates of the control cells (MCF-7aro) and all resistant cells were used for microarray experiments. Total of 23 samples were analyzed by microarray.

ORGANISM(S): Homo sapiens

SUBMITTER: Masri Selma

PROVIDER: S-ECPF-GEOD-17775 | biostudies-other |

REPOSITORIES: biostudies-other

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Project description:Resistance to endocrine therapy agents has presented a clinical obstacle in the treatment of hormone-dependent breast cancer. Our laboratory has initiated a study of microRNA regulation of signaling pathways that may result in breast cancer progression on aromatase inhibitors (AI). Microarray analysis of microRNA expression identified 115 significantly regulated microRNAs, of which 49 microRNAs were believed to be hormone-responsive. Within the AI-resistant cells, microRNAs were differentially expressed between the steroidal and non-steroidal AI-resistant lines. Also, a group of microRNAs were inversely expressed in the AI-resistant lines versus LTEDaro and tamoxifen-resistant. We focused our work on hsa-miR-128a which was hormone-responsive and up-regulated in the letrozole-resistant cell lines. Human miR-128a was shown to negatively target TGFBRI protein expression by binding to the 3âUTR region of the gene. Loss of TGFBRI resulted in compromised sensitivity to the growth inhibitory effects of TGFB in the letrozole-resistant lines. Inhibition of endogenous miR-128a resulted in re-sensitization of the letrozole-resistant lines to TGFB growth inhibitory effects. This data suggests that the hormone-responsive miR-128a can modulate TGFB signaling and survival of the letrozole-resistant cell lines. To our knowledge, this is the first study to address the role of microRNA regulation as well as TGFB signaling in AI-resistant breast cancer cell lines. We believe that in addition to estrogen-modulation of gene expression, hormone-regulated microRNAs may provide an additional level of post-transcriptional regulation of signaling pathways critically involved in breast cancer progression and AI-resistance. To look at microRNA expression profiles of breast cancer cell lines derived from MCF-7 cells that are resistant to endocrine therapy agents. MCF-7 cells that overexpress aromatase (MCF-7aro) were cultured long-term in the presence of endocrine therapy agents until cells acquired resistance. Three different aromatase inhibitors (letrozole, anastrozole or exemestane) were used, as well as the ER antagonist tamoxifen, or the hormone-free long-term estrogen deprived cells (LTED). Three replicates of the control cells (MCF-7aro) and all resistant cells were used for microarray experiments. Total of 23 samples were analyzed by microarray.

Project description:A functional genetic screen to identify genes causing tamoxifen resistance in an estrogen-dependent human breast cancer cell model was performed. By insertion of defective retrovirus into the genome, individual genetic changes were introduced at random in ZR-75-1 cells. Subsequently, infected cells were selected for their ability to proliferate while being exposed to tamoxifen, and from these cultures stable cell lines were established. The retrovirus insertion sites were mapped enabling the identification of several candidate breast cancer anti-estrogen resistance (BCAR) genes. By cDNA transfection of estrogen-dependent cells resulting in their transformation into a tamoxifen-resistant phenotype, proof was provided for the causative role of BCAR genes.A functional genetic screen to identify genes causing tamoxifen resistance in an estrogen-dependent human breast cancer cell model was performed. By insertion of defective retrovirus into the genome, individual genetic changes were introduced at random in ZR-75-1 cells. Subsequently, infected cells were selected for their ability to proliferate while being exposed to tamoxifen, and from these cultures stable cell lines were established. The retrovirus insertion sites were mapped enabling the identification of several candidate breast cancer anti-estrogen resistance (BCAR) genes identified. By cDNA transfection of estrogen-dependent cells resulting in their transformation into a tamoxifen-resistant phenotype, proof was provided for the causative role of BCAR genes (Van Agthoven et al, Breast Cancer Res Treat DOI:10.1007/s10549-008-9969-5, 2008). To elucidate the mechanisms how these individual BCAR genes induce cell proliferation in growth-arrested ZR-75-1 cells, we assessed the gene expression patterns between the different groups of cell lines. Keywords: Expression profiling, reference design RNA was isolated from two independent cultures of 69 tamoxifen-resistant cell lines. Detailed information regarding these cell lines with respect to the viral integration sites, was described previously (Van Agthoven et al, Breast Cancer Res Treat DOI:10.1007/s10549-008-9969-5, 2008). Each sample was hybridized in duplicate/triplicate and once in a dye-swap. The mixture of cell lines used as a reference was detailed previously (Jansen et al, J Clin Oncol 23, 732, 2005).

Project description:Introduction: Although changes in microRNA (miRNA) expression correlate with breast cancer diagnostic markers, comparatively little is known about miRNA regulation by selective estrogen receptor modulators (SERMs), e.g., tamoxifen (TAM), or their role in endocrine-resistance. Methods: A microarray approach was used to identify miRNAs differentially expressed and regulated by 4-hydroxyTAM (4-OHT) in MCF-7/TAM-sensitive versus LY2 TAM/endocrine-resistant human breast cancer cells after 6 h treatment. The expression of specific miRNAs was validated by quantitative, real-time PCR. Control miRNAs and time-course studies showed important gene-, time-, and cell- specific differences in miRNA expression. Results: 97 miRNAs were differentially expressed in MCF-7 versus LY2 cells. Bioinformatic analyses to impute the biological significance of these miRNAs by identifying 36 predicted gene targets of these miRNAs from amongst those reported to be regulated by MCF-7 cells was performed and agreement was found in direction of anticipated regulation for 12 of those putative targets. Among the miRNAs differentially expressed in MCF-7 versus LY2 cells, and that putatively target mRNAs regulated by 4-OHT in MCF-7 cells, are: miRs- 10a, 21, 22, 29a, 93, 125b, 181, 200a, 200b, 200c; 205, 221, and 222. Q-PCR assessment showed agreement with microarray data: miR- 10a, 22, 125b, 181, and 222 are higher in LY2 whereas miR-21 and miR-200a are lower in LY2 than MCF-7. 4-OHT increased miR-10a, miR-21, miR-22, miR-125b, miR-181a, miR-200a, and miR-222 in MCF-7 cells and reduced miR-10a in LY2 cells. E2 reduced the miR-21expression in MCF-7 cells. Treatment with ICI 182,780 revealed that ER suppresses miR-10a, miR-21, miR-22, miR-200a, miR-221, and miR-222 expression in MCF-7 cells. Time-dependent changes in select miRNA expression were observed in control, E2, and 4-OHT-treated MCF-7 cells. ESR1/ERα was significantly lower in LY2 than MCF-7, commensurate with higher let-7 family member, miR-221, and miR-222 expression in LY2. Reflecting inverse association with miR-200 family expression, the expression of ZEB1 was higher in LY2 than MCF-7 cells and concomitantly, E-cadherin expression was absent in LY2 cells indicating that LY2 have undergone epithelial to mesenchymal transition. Conclusions: Our studies identifying miRNAs with opposite expression between the two cell lines, indicate the involvement of these miRNAs in endocrine resistance. The purpose of this experiment is: 1. To identify miRNAs that are regulated (up or down) by 4-OHT in MCF-7 and LY2 cell lines (comparison of values from EtOH versus 4-OHT treated cells). 2. To identify miRNAs that are differentially regulated under basal (EtOH) conditions between MCF-7 and LY2 cell lines. 3. To identify miRNAs that are differentially regulated by 4-OHT in MCF-7 versus LY2 cells.

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MicroRNA expression profiles in Aromatase Inhibitor-Resistant, Tamoxifen-Resistant and LTED breast cancer cell lines

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